Luca Regni

The First Molecular Identification of an Olive Collection Applying Standard Simple Sequence Repeats and Novel Expressed Sequence Tag Markers

Germplasm collections of tree crop species represent fundamental tools for conservation of diversity and key steps for its characterization and evaluation. For the olive tree, several collections were created all over the world, but only few of them have been fully characterized and molecularly identified. The olive collection of Perugia University (UNIPG), established in the years’ 60, represents one of the first attempts to gather and safeguard olive diversity, keeping together cultivars from different countries. In the present study, a set of 370 olive trees previously uncharacterized was screened with 10 standard simple sequence repeats (SSRs) and nine new EST-SSR markers, to correctly and thoroughly identify all genotypes, verify their representativeness of the entire cultivated olive variation, and validate the effectiveness of new markers in comparison to standard genotyping tools. The SSR analysis revealed the presence of 59 genotypes, corresponding to 72 well known cultivars, 13 of them resulting exclusively present in this collection. The new EST-SSRs have shown values of diversity parameters quite similar to those of best standard SSRs. When compared to hundreds of Mediterranean cultivars, the UNIPG olive accessions were splitted into the three main populations (East, Center and West Mediterranean), confirming that the collection has a good representativeness of the entire olive variability. Furthermore, Bayesian analysis, performed on the 59 genotypes of the collection by the use of both sets of markers, have demonstrated their splitting into four clusters, with a well balanced membership obtained by EST respect to standard SSRs. The new OLEST (Olea expressed sequence tags) SSR markers resulted as effective as the best standard markers. The information obtained from this study represents a high valuable tool for ex situ conservation and management of olive genetic resources, useful to build a common database from worldwide olive cultivar collections, also based on recently developed markers.

Biofortification (Se): Does it increase the content of phenolic compounds in virgin olive oil (VOO)?

Extra-Virgin Olive Oil (EVOO) is a fundamental component of the Mediterranean diet and it may contain several anti-oxidant substances, such as phenols. Previous research has shown that this food may be enriched in phenols by spraying a sodium-selenate solution (100 mg L-1 Se) onto the crop canopy before flowering. The aim of this research was to evaluate the effect of this Se-fertilization before flowering (cv. Leccino) on the phenolic profile of EVOOs, and test to what extent such effects depend on the weather pattern, as observed in two contrasting experimental seasons (2013 and 2014). Results showed that Se-fertilisation enriched EVOOs both in selenium (up to 120 μg kg-1) and in phenols (up to 401 mg kg-1). This latter enrichment was related to an increase in PAL (L-Phenylalanine Ammonia-Lyase) activities and it was largely independent on the climatic pattern. Considering the phenolic profile, oleacein, ligustroside, aglycone and oleocanthal were the most affected compounds and were increased by 57, 50 and 32%, respectively. All these compounds, especially oleacein, have been shown to exert a relevant anti-oxidant activity, contributing both to the shelf-life of EVOOs and to positive effects on human health. It is suggested that Se-fertilisation of olive trees before flowering may be an interesting practice, particularly with poor cultivars and cold and rainy weather patterns, which would normally lead to the production of EVOOs with unfavourable phenolic profile.

Reuse of olive mill waste as soil amendment

The environmental impact of olive oil production is important as oil extraction can require a considerable amount of water and generates high quantities of olive mill wastes (OMW). In many olive-producer countries (e.g., Italy), OMW could be applied as amendment on the agricultural soils under specific requirements. Such application has raised interest not only because of its relatively low cost, but also for its potential to enhance soil fertility. Indeed, OMW high content in phosphorous, potassium, and organic matter could positively affect soil’s physicochemical characteristics improving crop productivity. However, OMW application to soils should be implemented with rationality since the high concentration of polymeric phenols have a well-known bacteriostatic effect on microorganisms and some phytotoxic effect on cultivations. In addition, a careful management of that application is required due to OMW acidic nature and high concentration in salts. In this chapter, the benefits and risks of using OMW as a soil amendment are discussed and recommendations on their proper application are also provided.

Selenium speciation profiles in biofortified sangiovese wine.

Biofortification is an agronomic-based strategy, utilized by farmers, to produce selenium (Se)-enriched food products that may help reduce dietary deficiencies of Se occurring throughout susceptible regions of the world. The foliar exposure route application ensures a high efficiency of Se assimilation by the plant since it does not depend on root-to-shoot translocation. In this study we treated grapevines of Sangiovese variety in the pre-flowering period with sodium selenate (100mg Se L-1). Se content was measured in leaves, fruit at harvest time and in wine respectively in treated and not treated samples with ICP-MS. At harvest, a higher amount of Se in the treated leaves compared to untreated ones was found, 16.0±3.1mgkg-1 dry weight (dw) against 0.17±0.006mgkg-1 dw in the untreated ones. The treated grapes had a content of Se of 0.800±0.08mgkg-1 dw, while that untreated one 0.065±0.025mgkg-1 dw. Immediately after the malolactic fermentation, the wine obtained from treated and untreated vines had a Se content of 0.620±0.09mg Se L-1 and 0.024±0.010mg Se L-1 respectively. In our case the percentage of inorganic Se is 26% of the total Se in the untreated wine, while in Se enriched wine this percentage increase to 47.5% of the total Se. The Se(VI) was the inorganic chemical form more present in enriched wine, probably due to foliar application with selenate. Distributions of Se species suggested being careful to the choice of the enrichment solutions to promote a balanced distribution of different chemical forms, perhaps favouring the accumulation of organic forms.

Long Term Amendment with Fresh and Composted Solid Olive Mill Waste on Olive Grove Affects Carbon Sequestration by Prunings, Fruits, and Soil

The soil amendment with organic wastes represents a way to increase the soil fertility and the organic carbon (C) stored in the agro-ecosystems. Among the organic waste materials produced by agricultural and industrial activities, olive mill wastes derived from the olive oil extraction process may represent a suitable soil amendment. The aim of the study was to evaluate the effect of fresh (SOMW) or composted mixture of SOMW and shredded olive tree prunings (C-SOMW+P) on the vegetative and productive activities of olive trees, on the C stored in the tree non-permanent structures (prunings and fruits) and in the soil. The plots treated with SOMW or C-SOMW+P showed higher vegetative and productive activities than the untreated plots, and this was attributed to the higher total N and availability of P and K supplied by the amendments. Consequently, treatments increased the C sequestered in the tree non-permanent structures than in the control trees. However, no significant different effect between SOMW and C-SOMW+P treatments was found for the C stored in prunings and fruits, whereas it was evident a stronger influence of C-SOMW+P than SOMW on soil C sequestration. Indeed, about 50% the C supplied by the treatment with C-SOMW+P was sequestered in the olive grove system, with more than 90% of the sequestered C stored into the soil. The low amount of C sequestered in the soil following the addition of SOMW was attributed to its richness of moisture and easily degradable compounds that triggered the mineralization processes controlled by the soil microbial community. Although the 8 years of amendment produced a higher fruit yields than the control, no difference occurred between the characteristics and the oil content of the olive fruits. Only the total phenol content for the oil obtained from the SOMW-treated plots was significantly higher. The other considered fruit characteristics did not show significant differences.

Use of Selenium-enriched olive leaves in the feed of growing rabbits: Effect on oxidative status, mineral profile and Selenium speciation of Longissimus dorsi meat

In the present study the use of Selenium-fortified olive leaves as potential dietary source of Se in rabbit nutrition was evaluated. Sixty New Zealand White rabbits (35 days of age) were randomly assigned to the following dietary treatments: standard diet (C), and C supplemented with either 10% olive leaves (OL) or 10% Selenium-fortified olive leaves (SeOL; 100 mg/L of foliar spray sodium selenate solution). At 70 days of age, 10 rabbits per group were slaughtered and the oxidative status, mineral profile and Selenium speciation of Longissimus dorsi meat was analyzed. Meat of the SeOL group exhibited better oxidative status (lower TBARS, higher GPx and α-tocopherol values) and a 5-fold higher Se content compared to that of the other treatments. The main Se form was SeMet (7-fold higher in the SeOL group), followed by SeCys2. The present trial demonstrates the possibility of using agro-industrial by-products as ingredients in rabbit feeds, thereby enriching meat bioactive compound content.

Selenium maintains Ca2+ homeostasis in sheep lymphocytes challenged by oxidative stress

Selenium (Se) is an essential element in human and animal diets, based upon a widespread range of beneficial effects that are primarily due to its antioxidant properties. While Se can be associated to anti-cancer and anti-diabetic activities, reproductive efficiency, and enhancement of the immune system, the mechanistic details of the corresponding biological processes are still largely elusive. To avoid deficiencies and increase bioavailability, Se it is generally supplied to livestock through Se-supplemented feeds or forage plants fertilized with inorganic Se. While the relationship between Ca2+ and ROS (reactive oxygen species) is well known, only a few studies have addressed the possible involvement of Se in the control of cytosolic Ca2+ in oxidative stress. The results on Ca2+ homeostasis were obtained adding exogenous Se in the form of SeO42- to sheep lymphomonocytes cultured in vitro. In particular, Se strongly attenuated 1mM H2O2-induced alteration of intracellular [Ca2+]C as well as the entry of extracellular Ca2+ into the cells with comparable EC50 values for sodium selenate accounting to 1.72 and 2.28 mM, respectively. In an ex vivo trial, it was observed that Ca2+ homeostasis can effectively be rescued in sheep lymphomonocytes exposed in vivo to a Se concentration of approximately 1.9 mM, that was achieved by feeding sheep with olive leaves previously sprayed with 500 mg/plant Na-selenate. Thus the results obtained suggest that the mode of action of selenium markedly influenced Ca2+-related signaling events. Furthermore, results clearly reveal that the protective effect of Se on Ca2+ homeostasis under oxidative challenge can be clearly and effectively achieved through an appropriate dietary regimen obtained also in a circular economy logic using pruning of olive trees treated to reduce tree drought stress.

Use of olive leaves (whether or not fortified with sodium selenate) in rabbit feeding: Effect on performance, carcass and meat characteristics, and estimated indexes of fatty acid metabolism

Sixty New Zealand White weaned rabbits were divided into three groups and subjected to different dietary treatments: a standard diet for the control (C), a standard feed supplemented with 10% of plain olive leaves (OL) and a standard feed supplemented with 10% of selenium-fortified olive leaves (100 mg/L of foliar spray sodium selenate solution; SeOL). The productive performance was recorded at the time of slaughter (after 35 days); the carcass and meat traits were determined and estimated indexes of fatty acid metabolism were calculated. No significant differences were found on the rabbit productive performance and the physical-chemical characteristics of the meat. Both group of rabbits on the enriched diet showed leaner and thinner carcasses and a higher meat concentration of oleic acid. The estimated index of Δ5 + Δ6-desaturase, starting from n-6 fatty acids, was lower in both groups supplemented with leaves. The use of selenium-fortified olive leaves, positively affected the lipid oxidative stability of rabbit meat.

Determination of changes in the concentration and distribution of elements within olive drupes (cv. Leccino) from Se biofortified plants, using laser ablation inductively coupled plasma mass spectrometry: Concentrations and distribution of Selenium and other elements in Se-biofortified olive

BACKGROUND Biofortification of food crops has been used to increase the intake of Se in the human diet, even though this may change the concentration of other elements and modify the nutritional properties of the enriched food. Selenium biofortification programs should include routine assessment of the overall mineral composition of enriched plants. RESULTS Laser ablation inductively coupled plasma mass spectrometry (LA ICP-MS) was used for the assessment of mineral composition of table olives. Olive trees were fertilized with sodium selenate before flowering. At harvest, the edible parts of drupes proved to be significantly enriched in Se, delivering 6.1 μg g-1 (39% of the RDA for five olives). Such enrichment was followed by significant changes in the concentrations of B, Mg, K, Cr, Mn, Fe and Cu in edible parts, which are discussed for their impact on food quality. CONCLUSION The biofortification of olive plants has allowed the enrichment of fruits with selenium. Enrichment with selenium has caused an increase in the concentration of other elements, which can change the nutritional quality of the drupes. The analytical technique used well as a valuable tool for routinely determining the chemical composition of all fruit parts.

The Selenium Supplementation Influences Olive Tree Production and Oil Stability Against Oxidation and Can Alleviate the Water Deficiency Effects

Foliar fertilization with selenium (Se) may well be beneficial in increasing the nutritional and qualitative values of food in Se-deficient regions such as the Mediterranean Basin, and may contribute to an increase in drought resistance in plants. The present study has considered detachment force, flesh firmness, pigmentation, fresh and dry weight, and oil content of olive drupes from Se fertilized olive orchards (Olea europaea L.) under drought stress and well-watered conditions. This study has also evaluated the total Se, Se amino acid, phenol, carotenoid and chlorophyll contents of EVOO, plus its oxidative stability against oxidation. While there was no change in the ripening indexes and the production of olives generally, Se application did increase the total Se, Se methionine, phenol, and carotenoid and chlorophyll contents. The higher concentration of these (bio) chemical compounds in EVOO obtained from Se fertilized plants might well suggest enhanced antioxidant activity. Consequently, EVOO obtained from Se fertilized trees possesses a higher nutritional value and, as indicated by the greater oxidative stability against oxidation, longer shelf life. In addition, under water deficient conditions, a higher fresh olive weight corresponds to a higher level of phenol, carotenoid and chlorophyll, and the chlorophyll-to-carotenoid ratio in Se fertilized trees would appear to confirm the positive role of selenium in alleviating damage caused by drought stress conditions.